Ian Mullaney

Identification of Two Distinct Isoforms of the Guanine Nucleotide Binding Protein Go in Neuroblastoma × Glioma Hybrid Cells: Independent Regulation During Cyclic AMP‐Induced Differentiation

Abstract: Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the α‐subunit of the (pertussis toxin‐sensitive) guanine nucleotide binding protein Go were used in two‐dimensional immunoblots of membranes of neuroblastoma × glioma (NG108–15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108–15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8‐bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of Goα, as has previously been noted in onedimensional immunoblots. Two‐dimensional analysis demonstrated that the cAMP‐induced increases in levels of Goα were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin‐catalysed ADP‐ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of Goα could be achieved in one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of Go from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of Go from the cells. In agreement with the data from two‐dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP‐induced differentiation of NG108–15 cells. Of these two forms of “Go,” the acidic species is equivalent to Go from brain, but the basic form is not identical with Go, which has been purified from bovine brain.

Regulation of Spontaneous Activity of the δ‐Opioid Receptor: Studies of Inverse Agonism in Intact Cells

Abstract: Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [3H]adenine in intact Rat‐1 fibroblasts that had been stably transfected to express the murine δ‐opioid receptor (clone D2). Basal [3H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin d‐Ala2,d‐Leu5 enkephalin (DADLE) produced strong inhibition of forskolin‐amplified [3H]cyclic AMP production, whereas the δ‐opioid ligand ICI174864 augmented forskolin‐amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC50 (9.4 ± 0.6 × 10−8M) for ICI174864 augmentation of forskolin‐stimulated adenylyl cyclase was equal to its estimated Ki. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.

Distribution and relative levels of expression of the phosphoinositidase-C-linked G-proteins Gqα and G11α : absence of G11α in human platelets and haemopoietically derived cell lines

Abstract Membranes of a variety of clonal cell lines, including neuroblastoma× glioma hybrid NG108-15, glioma C6, Rat 1 and CHO fibroblasts and the pituitary-derived cell lines αT3 and GH3 were immunoblotted with an antiserum (CQ) raised against a synthetic peptide corresponding to the C-terminal decapeptide of the α subunits of the phosphoinositidase-C-linked G-proteins Gq and G11. In SDS-PAGE conditions able to resolve these two polypeptides, direct evidence was obtained for co-expression of these two G-proteins in all of the above cell lines. The ratio of these two G-proteins varied substantially ( α 11 α q = 0.25–2.5 ) between the cell lines. In human platelets and in a range of haemopoietically derived human cell lines including U937 (monoblasts), Raji (Burkitts lymphoma) and Jurkat (mature T cell) expression of G11α was not detected. This was not due to the inability of the antiserum to identify human G11α as other human cell lines co-expressed both G-proteins. A third, unidentified CQ reactive polypeptide of similar mobility was resolved and present in all cell lines examined.

The human muscarinic M1 acetylcholine receptor, when expressed in CHO cells, activates and downregulates both Gqα and G11α equally and non-selectively

CHO cells express both of the phosphoinositidase C‐linked G‐proteins Gq and G11 G11α is some 2.5‐fold more highly expressed than Gqα in membranes of these cells. Following transfection and stable expression of CHO cells with DNA encoding the human muscarinic M1 acetylcholine (HM1) receptor, chronic treatment of the cells with the cholinergic agonist carbachol resulted in down‐regulation of membrane levels of both Gqα and G11α. Dose‐response curves to carbachol produced identical EC50 values for agonist‐induced down‐regulation of the two G‐proteins and both were down‐regulated with the same time course. These data indicate that the HM1 receptor interacts with and activates both Gqα and G11α equivalently and non‐selectively in a whole cell system in which the receptor has access to both G‐proteins.

GTP-Binding proteins in brain and neutrophil are tethered to the plasma membrane via their amino termini

Using specific antisera raised against synthetic peptides, we find that three distinct GTP-binding protein alpha subunits remain bound to the plasma membrane even after activation with nonhydrolyzable GTP analog. Trypsin cleaves each alpha subunit at a site near the amino-terminus, and quantitatively releases the large fragment (comprising all but an amino-terminal 2 kDa piece) from the membrane. Our results indicate that alpha subunits are essentially cytoplasmic proteins tethered to the inner surface of the membrane via an amino terminal stalk.

GTP analogues cause release of the alpha subunit of the GTP binding protein, GO, from the plasma membrane of NG108-15 cells

Incubation of membranes of neuroblastoma x glioma hybrid, NG108-15 cells with GDP beta S followed by immunoblotting of resolved membrane and supernatant fractions with specific anti-peptide antisera showed essentially all of the alpha subunit of Go to be associated with the membrane. Similar experiments with poorly hydrolyzed analogues of GTP caused release of a significant fraction (some 50% within 60 minutes) of Go alpha into the supernatant. This was not mimicked by analogues of ATP. Antisera directed against peptides corresponding to the extreme N and C-termini of GO alpha demonstrated that the released polypeptide was not proteolytically clipped. These experiments show that the alpha subunit of GO need not be invariably bound to the plasma membrane and that guanine nucleotide activation can release the alpha subunit of GO from its site of membrane attachment.

Elevated levels of the guanine nucleotide binding protein, Go, are associated with differentiation of neuroblastoma × glioma hybrid cells

Each of a range of pharmacological agents which function to increase intracellular levels of cAMP caused a morphological ‘differentiation’ of neuroblastoma × glioma hybrid, NG108‐15, cells grown in tissue culture. Associated with this differentiation, increased incorporation of [32P]ADP‐ribose catalysed by pertussis toxin was noted into a band of some 39–40 kDa in membranes derived from these cells. Immunoblotting using two antipeptide antisera which identify different regions of Goα demonstrated marked increases in the levels of this polypeptide in membranes of the differentiated cells. However, levels of the β‐subunit did not increase appreciably with differentiation.

Immunological identification of the α subunit of G13, a novel guanine nucleotide binding protein

An antiserum (13CB) was generated against a synthetic peptide, HDNLKQLMLQ, which is predicted to represent the C‐terminal decapeptide of the α subunit of the novel G‐protein, G13. Competitive ELISA indicated that the antiserum reacted with this peptide but that it showed minimal ability to recognize peptides which represent the equivalent regions of the pertussis toxin‐insensitive G‐proteins, Gq + G11, G12, G15 + G16, GL1 (also called G14) as Gz, and well as other G‐proteins. Immunoblots of human platelet membranes with antiserum 13CB identified a single 43‐kDa polypeptide, and while this immunoreactivity was abolished by the presence of the cognate peptide it was not modified by the presence of peptides corresponding to the equivalent region of other G‐proteins. Immunoreactivity corresponding to G13α was detected in a range of cell types with human platelets having the highest levels of this polypeptide.

Expression of the Human β2‐Adrenoceptor in NCB20 Cells Results in Agonist Activation of Adenylyl Cyclase and Agonist‐Mediated Selective Down‐Regulation of Gsα

Abstract: Murine neuroblastoma × embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human β2‐adrenoceptor under the control of a β‐actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the β‐adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild‐type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein α subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane‐associated levels of Gsα in membranes of clone L9 cells and a 50% reduction in Gsα levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gsα in wild‐type NCB20 cells, and such treatment had no effect on the levels of other G protein α subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half‐maximal loss of Gsα in clone D1 was achieved within 1–2 h of addition of agonist. Dose‐response curves to isoprenaline in clone D1 indicated that half‐maximal down‐regulation of Gsα was produced by ∼1 nM agonist. Measurement of Gs mRNA levels in both clones, however, using both reverse transcriptase‐polymerase chain reaction and northern blotting revealed no significant change following treatment with isoprenaline.

Overexpression of Gsα in NG108-15, neuroblastoma X glioma cells: effects on receptor regulation of the stimulatory adenylyl cyclase cascade

Neuroblastoma X glioma hybrid, NG108‐15 cells were stably transfected with an epitope tagged variant of Gsα(HAGsα). The introduced HA‐Gsα was able to interact with the IP prostanoid receptor and was able to stimulate adenylyl cyclase activity as measured by an enhanced capacity of membrane extracts to reconstitute NaF‐dependent adenylyl cyclase activity to membranes of S49 lymphoma cyc− cells. Despite this, neither the maximal stimulation nor the potency of agonist ligands at the IP prostanoid, A2 adenosine or secretin receptors was altered substantially compared to the parental cells although the basal adenylyl cyclase activity was increased. These data indicate that cellular levels of Gsα do not limit signal transduction capacity in NG108‐15 cells, whereas enhanced expression of adenylyl cyclase allows greater maximal cAMP generation following receptor activation (MacEwan, D.J., Kim, G.D. and Milligan, G. (1996) Biochem. J. 318, 1033–1039).